Lecture 6: Methods 2

Question 1

What is something the conventional crystalography misses? What could we then find out specifically?

Answer 1

We cannot capture the dynamics of the proteins - it basically takes just an image of it -> so we cannot infer anything about its actions
- that we could have insignts into catalytic actions, isomerazation, side chain motions, enzymatic reactions
- each of these changes have its duration in which it happens (e.g. some extremely quick)

 - E.g. how exactly does retinal changes upon light (in femtoseconds), water splitting in photosystem 2, photoactive yellow protein (used in optogenetics)

Question 2

Give an example of a protein that could be studies via Cryotrapping?

Answer 2

• Orange Carotenoid Protein (OCP)
  = light-harvesting controller of cyanobacteria
  
  • Has 2 subunits: C-term, N-term + in between keratinoid molecule -> how does it absorbe light and triggers structural changes
    - OCP changes its color upon activation (e.g. blue light -> absorbed -> enters photocycle -> turns red)
    - tricky to study because simply shining light won’t make it go into photocycle

Question 3

How was the example resolved? How would you define cryotrapping?

Answer 3

Illuminating single OCP crystals at different timepoints -> resolve structural intermediate
- isomerazation occurs (transient process) triggering conformational changes

• Cryotrapping = collecting diffraction data from crystals at high-intensity X-ray sources by cooling them to form amorphous ice, which helps in preserving the crystal structure for analysis -> doing so at different timepoints

Question 4

Look at another example:

Answer 4

Resolving structural intermediates of a ligand binding to a receptor (ligand was introduced and frozen at varying times)

Question 5

What is are disadvantages of cryotrapping? What can we do about it?

Answer 5

• we cannot study processes that are too fast - only things occuring e.g. within seconds
• forced to investigate in freezing temperature with the assumption that proteins will act the same -> not always the case

=> We can choose a more appropriate method e.g. Laue

Question 6

How does Laue differ from normal crystalography?

Answer 6

Normal crystalography uses monochromatic beams (one type of X-ray wavelength) X Laueutilizes polychromatic approach (pink beam)
-> we get more Bragg spots (more situations atwhich Braggs law is met)
-> we need less data (there is enough to get rid of the noise)

=> resolve structural changes at different time delayes
- e.g. yellow protein